Beta-glucan compounds, compositions, and methods

ABSTRACT

Described herein are beta-glucan compounds, compositions, and methods. Generally, the methods exploit the observation that beta-glucan compounds can bind to B cells. Thus, the methods generally include administering a beta-glucan compound to a subject in an amount effective for the beta-glucan compound to bind to a B cell and modulate at least one biological function of the B cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 14/116,550, now U.S. Pat. No. 9,934,607, filed Apr. 16, 2014, whichis a U.S. National Stage Application of International Application No.PCT/US2012/037073, entitled (3-GLUCAN COMPOUNDS, COMPOSITIONS, ANDMETHODS, filed on May 9, 2012, which claims priority to U.S. ProvisionalPatent Application Ser. No. 61/483,983, filed May 9, 2011, each of whichis incorporated by reference herein in its entirety.

BACKGROUND

IMPRIME PGG(β(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose,Biothera, Eagan, Minn.) is a soluble form of yeast-derived β-glucan.Laminarin is another example of a β-1,3/1,6 glucan, however laminarin isderived from algae and differs chemically from IMPRIME PGG.

Yeast β-glucans are conserved microbial structures not found inmammalian cells. IMPRIME PGG is a soluble β-glucan isolated from yeast.Since IMPRIME PGG is a conserved microbial structure not found inmammals, and IMPRIME PGG is recognized by innate immune cells, IMPRIMEPGG is classified as a pathogen-associated molecular pattern (PAMP). Ingeneral, PAMPs are microbial components that are first recognized by theinnate immune system resulting in immune activation.

Activation of the innate immune system with soluble β-glucans can resultin anti-tumor activity in mice (Allendorf et al., 2005, J Immunol.,174(11):7050-7056; Li et al., 2006, J Immunol., 177(3):1661-1669;Salvador et al., 2008, Clinical Cancer Research, 14:1239-1247). Unlikeother PAMPs (e.g., LPS, Pam3Cys, poly I:C), IMPRIME PGG does not appearto induce overt production of pro-inflammatory cytokines such as, forexample, the NF-κB-regulated cytokines tumor necrosis factor-α (TNF-α)and interferons (e.g., IFN-α and IFN-γ). Thus, IMPRIME PGG may modulateimmune responses in a different manner than other PAMPs.

SUMMARY

In one aspect, this disclosure describes a method that generallyincludes administering to a subject an amount of a β-glucan compoundeffective to bind to B cells and modulate at least one biologicalfunction of the B cells.

In some embodiments, the β-glucan compound generally includes a β-glucanmoiety and an active moiety. In some such embodiments, the active moietycan include an immunomodulator, an antibody, an antigen, a cytotoxicagent, a cytokine, an inhibitor of Bcl-2, a kinase inhibitor, an mTORinhibitor, a proteosome inhibitor, or an immunosuppressive agent. Insome such embodiments, the β-glucan moiety and the active moiety may becoupled through a covalent linkage. In other such embodiments, theβ-glucan moiety and the active moiety may be coupled through an affinitylinkage.

In some embodiments, the β-glucan can be a soluble β-glucan, while inother embodiments the β-glucan can be a particulate β-glucan. In someembodiments, the β-glucan compound can include a derivatized β-glucan ora modified β-glucan.

In some embodiments, the biological function of the B cells comprisesmaking an immunoglobulin.

In some embodiments, modulating at least one B cell biological functioncomprises killing B cells.

In another aspect, the invention provides a β-glucan compound thatgenerally includes a β-glucan moiety coupled to an active moiety.

In some embodiments, the active moiety can include an immunomodulator,an antibody, an antigen, a cytotoxic agent, a cytokine, or animmunosuppressive agent. In some of these embodiments, the β-glucanmoiety and the active moiety may be coupled through a covalent linkage.In other such embodiments, the β-glucan moiety and the active moiety maybe coupled through an affinity linkage.

In some embodiments, the β-glucan moiety is derived from yeast. In someof these embodiments, the yeast can include Saccharomyces cerevisiae. Inone particular embodiment, the β-glucan moiety can includer3(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose.

In another aspect, this disclosure describes a pharmaceuticalcomposition that generally includes a β-glucan compound as describedherein.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Truncated structure ofβ(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose.

FIG. 2A. IMPRIME PGG binding to B cells in whole blood.

FIG. 2B. IMPRIME PGG binding to B cells in PBMCs.

FIG. 2C. Laminarin binding to B cells in PBMCs.

FIG. 3A. Surface CR2 expression on PBMC subsets (shaded peak−No IMPRIMEPGG; open peaks−MPRIME PGG+Isotype ctrl and IMPRIME PGG+anti-CR2blocking antibody).

FIG. 3B. Blocking of IMPRIME PGG binding on B cells in whole blood byanti-CR2 antibody (shaded peak−No IMPRIME PGG; open peaks−IMPRIMEPGG+Isotype ctrl and IMPRIME PGG+anti-CR2 blocking antibody).

FIG. 3C. Blocking of IMPRIME PGG binding on B cells in PBMCs by anti-CR2antibody (shaded peak−No IMPRIME PGG; open peaks−IMPRIME PGG+Isotypectrl and IMPRIME PGG+anti-CR2 blocking antibody).

FIG. 4A. CR2 surface expression on human Daudi and Raji B cell tumorcell lines (shaded peak−isotype; open peak−anti-CR2) (shaded peak−NoIMPRIME PGG; open peaks−50 mcg/mL IMPRIME PGG and 200 mcg/mL IMPRIMEPGG).

FIG. 4B. Binding of IMPRIME PGG to Daudi and Raji B cell tumor celllines (shaded peak−No IMPRIME PGG; open peaks−50 mcg/mL IMPRIME PGG and200 mcg/mL IMPRIME PGG).

FIG. 5. IMPRIME PGG vs. BSA-conjugated IMPRIME PGG (BT-1110) binding tohuman B cells in whole blood.

FIG. 6. IMPRIME PGG vs. Benzyl-amine derivatized IMPRIME PGG (BT-1222)binding to human B cells in whole blood.

FIG. 7. Targeted binding of (3 glucan (IMPRIME PGG) and β-glucanconjugate to B cells. (A) β-glucan (IMPRIME PGG-treated) and βglucan-ERBITUX conjugate (ERBITUX-IMPRIME PGG conjugated-treated) bindto B cells and are detected using an anti-β-glucan antibody. (B)β-glucan-ERBITUX conjugate binds to B cells and is detected using ananti-Human IgG antibody.

FIG. 8. Calcium flux in cells treated in vitro with soluble β-glucan,then stimulated with IgM.

FIG. 9. Calcium flux in cells of subjects treated in vivo withparticulate β-glucan, then stimulated with IgM.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention generally involves binding of β-glucan compounds to Bcells, including B cell tumors, and methods that exploit this bindingactivity. As a result, β-glucan compounds may be used as a modulator ofB cell function. Thus, β-glucan compounds may be used to target B cellsin circumstances in which it may be desired to activate one or more Bcell functions such as, for example, the production of a neutralizing orgrowth-inhibiting antibody. This may be useful for prophylactic ortherapeutic treatments of, for example, infectious and/or neoplasticconditions. Alternatively, β-glucan compounds may be used to target Bcells in circumstances in which it may be desirable to inhibit B cellfunctions. This may be useful for treatments of, for example, B cellneoplasms such as, for example, B cell chronic lymphocytic leukemia, orautoimmune conditions such as, for example, any condition in which onecomponent of the condition involves dysregulation of antibody productionsuch as, for example, Lupus or rheumatoid arthritis.

Thus, in one aspect, the invention provides methods that generallyinclude administering to a subject an amount of a β-glucan compoundeffective to bind to B cells and modulate at least one biologicalfunction of the B cells.

The β-glucan compound or as in certain embodiments discussed in moredetail below, a β-glucan moiety of a β-glucan compound can be or bederived from, for example, β-glucan derived from a fungal yeast sourcesuch as, for example, Saccharomyces cerevisiae, Torula (candida utilis),Candida albicans, Pichia stipitis, or any other yeast source; β-glucanderived from another fungal source such as, for example, scleroglucanfrom Sclerotium rofsii or any other non-yeast fungal source; β-glucanfrom an algal source such as, for example, laminarin or phycarine fromLaminaria digitata or any other algal source; β-glucan from a bacterialsource such as, for example, curdlan from Alcaligenes faecalis or anyother bacterial source; β-glucan from a mushroom source such as, forexample, schizophyllan from Schizophyllan commune, lentinan fromLentinan edodes, grifolan from Grifola frondosa, ganoderan fromGanoderma lucidum, krestin from Coriolus versicolor, pachyman from Poriacocos Wolf or any other mushroom source; β-glucan derived from a cerealgrain source such as, for example, oat glucan, barley glucan, or anyother cereal grain source; β-glucan derived from a lichen source suchas, for example, pustulan from Umbilicaris pustulata, lichenan fromCetraria islandica, or any other lichen source. The form of glucan usedto make these conjugates can be either soluble or insoluble in water. Insome embodiments, the β-glucan or β-glucan moiety is water-soluble.

In some embodiments, the β-glucan or β-glucan moiety may be, or bederived from Saccharomyces cerevisiae. One such form of β-glucan derivedfrom Saccharomyces cerevisiae isβ(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose.β(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose can beprovided in various forms. One form of this β-glucan is a particulateform described, for example, in U.S. Pat. No. 7,981,447. The β-glucancan form particles ranging in size from a minimum of 0.1 μM to about 6.0μM. In some cases, the particles are not water-soluble. A water-solubleform of thisβ(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-b(1,3)-D-glucopyranose isreferred to herein as IMPRIME PGG and is described in, for example, U.S.Patent Application Publication No. US2008/0103112 A1. Laminarin isanother example of a β-1,3/1,6 glucan. Laminarin, however, is derivedfrom algae and differs chemically from the particulate and IMPRIME PGGforms of yeast β-glucan. Some of those differences are reflected inTable 1.

TABLE 1 Compound IMPRIME PGG Laminarin Biological source Saccharomycescerevisiae Laminaria digitata (yeast) (algae) Average Mol. Wt. 150,0008,000 % Branching 4% 8%

In some embodiments, the β-glucan compound can include a β-glucan moietycoupled to an active moiety. As used herein, the term “moiety” andvariations thereof refer to a portion of a chemical compound thatexhibits a particular character such as, for example, a particularbiological or chemical function such as, for example, immunomodulation,cytotoxicity, solubility, bioavailability, metabolism and/or targetspecificity.

The active moiety may include any compound that possesses a particularactivity toward B cells. The activity may, for example, includemodulating one or more B cell biological functions or cytotoxicactivity. As used herein, “modulate” and variations thereof refer to asubstantial increase or decrease in biological function. A substantialincrease or decrease in biological activity is an increase or decreasebeyond a predetermined threshold increase or decrease in the biologicalfunction. Thus, the active moiety may induce or, alternatively, inhibitone or more B cell biological functions. As used herein, “induce” andvariations thereof refer to any measurable increase in biologicalfunction. For example, induction of B cells may be reflected by, forexample, inducing the B cells to produce an antigen-specific antibody.“Inhibit” and variations thereof refer to any measurable reduction ofbiological function. For example, inhibition of B cells can include, forexample, causing the B cells to produce less antigen-specific antibodythan in the absence of the inhibitory stimulus. The extent of inhibitionmay be characterized as a percentage of a normal level of activity.“Biological function” refers to cellular activity (e.g., antibodyproduction, cytokine production, surface receptor modulation, cellularproliferation) that is characteristic of an identified cell type.

The active moiety may be, or be derived from, an adjuvant orimmunomodulator. Exemplary immunomodulators include, for example,pathogen-associated molecular patterns (PAMPs) and/or danger-associatedmolecular patterns (DAMPs).

PAMPs include molecules that are often associated with groups ofpathogens and are recognized by cells of the innate immune system. PAMPscan be referred to as small molecular motifs conserved within a class ofmicrobes. They are recognized by Toll-like receptors (TLRs) and otherpattern recognition receptors (PRRs). PAMPs can activate innate immuneresponses by identifying some conserved non-self molecules. Bacteriallipopolysaccharide (LPS), an endotoxin found on the bacterial cellmembrane of many bacteria, is an exemplary PAMP. LPS is specificallyrecognized by TLR 4, a recognition receptor of the innate immune system.Other PAMPs include, for example, bacterial flagellin (recognized by TLR5), lipoteichoic acid from Gram positive bacteria, peptidoglycan, andnucleic acid variants normally associated with viruses, such as, forexample, double-stranded RNA (dsRNA, recognized by TLR 3), unmethylatedCpG motifs (recognized by TLR 9), or certain imidazoquinoline aminederivatives that are recognized by TLR 7 and/or TLR 8.

DAMPs include molecules that can initiate and perpetuate immune responsein the noninfectious inflammatory response. Many DAMPs are nuclear orcytosolic proteins with defined intracellular functions that, whenreleased outside the cell following tissue injury, move from a reducingto an oxidizing milieu resulting in their functional denaturation. Also,following necrosis, tumor DNA may be released into the extranuclearspace/extracellular micro-environment and functions as a DAMP. DAMPs canvary greatly depending on the type of cell (e.g., epithelial versusmesenchymal) and injured tissue. Protein DAMPs include, for example,intracellular proteins such as, for example, heat-shock proteins orHMGB1 (high-mobility group box 1), and proteins derived from theextracellular matrix that are generated following tissue injury, such ashyaluronan fragments. Examples of non-protein DAMPs include ATP, uricacid, heparin sulfate, and DNA.

Thus, in some embodiments, the active moiety may be, or be derived froma PAMP or a DAMP such as, for example, an agonist of one or moreToll-like receptors (TLRs). In some embodiments, the active moiety maybe an agonist of TLR 1 (e.g., a triacyl lipopeptide), an agonist of TLR2 (e.g. lipoteichoic acid), an agonist of TLR 3 (e.g., dsRNA), anagonist of TLR 4 (e.g., lipopolysaccharide), an agonist of TLR 5 (e.g.,flagellin), an agonist of TLR 6 (e.g., peptidoglycan), an agonist of TLR7 (e.g., ssRNA, imidazoquinolines, loxoribine), an agonist of TLR 8(e.g., imidazoquinolines, loxoribine), or an agonist of TLR 9 (e.g., anunmethylated CpG oligonucleotide).

In other embodiments, the active moiety can include, or be derived froman antibody. Certain antibodies are known to activate B cells and,therefore, induce B cells biological functions. Such an antibody caninclude or be derived from, for example, an anti-CD20 antibody (e.g.,rituximab), an anti-CD22 antibody (e.g., epratuzumab), an anti-CD70antibody, an anti-CD40 antibody, or an anti-CD137 antibody. CD137 isalso known as 4-1BB, so an anti-CD137 antibody may alternatively bereferred to as an anti-4-1BB antibody. An active moiety may include animmunologically active fragment of an antibody such as, for example, ascFv, a Fab, a F(ab′)₂, a Fv, or other modified antibody fragment.

In other embodiments, the active moiety can include an antigen. As usedherein, “antigen” and variations thereof refer to any material capableof raising an immune response in a subject challenged with the material.In various embodiments, an antigen may raise a cell-mediated immuneresponse, a humoral immune response, or both. Suitable antigens may besynthetic or occur naturally and, when they occur naturally, may beendogenous (e.g., a self-antigen) or exogenous. Suitable antigenicmaterials include but are not limited to peptides or polypeptides;lipids; glycolipids; polysaccharides; carbohydrates; polynucleotides;prions; live or inactivated bacteria, viruses, fungi, or parasites; andbacterial, viral, fungal, protozoal, tumor-derived, or organism-derivedimmunogens, toxins or toxoids.

Thus, exemplary antigens include viral antigens such as, for example,antigens associated with influenza, Hepatitis A, Hepatitis B, HepatitisC, adenovirus, Herpes Simplex B, or other suitable virus.

Exemplary antigens also can include an antigen associated with aparticular type of neoplasm or tumor. Such antigens include, forexample, MUC-1, CA 125, telomerase/hTERT, PSA, NY-ESO-1, MAGE, AML1fusions, EGFR, HER2/NEU, gp100, WT1, CEA, or other antigen having aknown association with one or more tumors.

Exemplary antigens also can include bacterial antigens such as, forexample, tetanus toxoid, diphtheria toxoid, a Staphylococcus spp.antigen, a Pneumococcus spp. antigen, a Klebsiella spp. antigen, oranother bacterial antigen.

Exemplary antigens also can include parasitic antigens such as, forexample, a Trypanosoma spp. antigen, a Toxoplasma spp. antigen, aLeishmania spp. antigen, a Plasmodium spp. antigen, a Schistosoma spp.antigen, or another parasitic antigen.

In other embodiments, the active moiety can include, or be derived from,a cytotoxic agent. Such embodiments may have particular utility wherethe B cell may be targeted for killing such as, for example, in the caseof certain B cell lymphomas. Thus, the targeted delivery of such agentscan permit systemic delivery while reducing the likelihood, extent,and/or severity of systemic side effects. Cytotoxic agents can include,for example, chemotherapeutic agents such as, for example, cisplatin,fludarabine, cyclophosphamide, doxorubicin, vincristine, carboplatin,ifosfamide, etoposide, cytarabine, paclitaxel, or ABRAXANE (CelgeneCorp., Summit, N.J.). Other exemplary cytotoxic agents include, forexample, ricin A chain or diphtheria toxin. Cytotoxic agents also caninclude certain radioactive isotopes such as, for example, yttrium-90 oriodine-131.

In some embodiments, the active moiety can include a cytokine such as,for example, IL-10, IL-12, or recombinant forms thereof.

In other embodiments, the active moiety can be, or be derived from,immunosuppressive agents. Exemplary immunosuppressive agents include,for example, a corticosteroid, tacrolimus, or methotrexate.

In other embodiments, the active moiety can be, or be derived from,inhibitors of the Bcl-2 family of proteins. Exemplary inhibitorsinclude, for example, small molecules, antisense oligonucleotides, orBcl-2 homology 3 (BH3) mimetic peptides.

In other embodiments, the active moiety can be, or be derived from,small molecule inhibitors of kinases that modulate B cell function.Exemplary examples of kinases include, for example, Bruton's tyrosinekinase (Btk), spleen tyrosine kinase (Syk), or phosphoinositide-3 kinase(PI3K).

In other embodiments, the active moiety can be, or be derived from,small molecule inhibitors of other targeted kinases for oncology.Exemplary examples of targeted kinases include, for example, Bcr-Abl,PDGFR, c-KIT, DDR1, EGFR, ERBB2 (HER2), HER4, VEGFR, VEGFR (b-raf), SRCfamily, or TEC family. Exemplary examples of approved kinase inhibitorsthat could be the active moiety, or from which the active moiety may bederived, include, for example, imatinib (e.g., GLEEVEC, NovartisPharmaceuticals Corp., East Hanover, N.J.), nilotinib (e.g., TASIGNA,Novartis Pharmaceuticals Corp., East Hanover, N.J.), erlotinib (e.g.,TARCEVA, Genentech, Inc., South San Francisco, Calif.), gefitinib (e.g.,IRESSA, AstraZeneca Pharmaceuticals LP, Wilmington, Del.), sorafenib(e.g., NEXAVAR, Onyx Pharmaceuticals, Inc., South San Francisco,Calif.), sunitinib (e.g., SUTENT, Pfizer, Inc., New York, N.Y.),lapatinib (e.g., TYKERB, GlaxoSmithKline plc, Philadelphia, Pa.), anddasatinib (e.g., SPRYCEL, Bristol-Myers Squibb, Princeton, N.J.).

In other embodiments, the active moiety can be, or be derived from, mTOR(mammalian target of rapamycin) inhibitors (e.g., approved mTORinhibitors include everolimus (e.g., AFINITOR and ZORTRESS, NovartisPharmaceuticals Corp., East Hanover, N.J.) and temsirolimus (TORISEL,Pfizer, Inc., New York, N.Y.).

In other embodiments, the active moiety can be, or be derived from,proteasome inhibitors that inhibit the NF-κB pathway such as, forexample, bortezomib (e.g., VELCADE, Millennium Pharmaceuticals, Inc.,Cambridge, Mass.)

In some embodiments, the β-gluc an moiety may be coupled to the activemoiety. The β-gluc an moiety and the active moiety may be covalentlycoupled or, in some embodiments, may include at least one affinity orionic bond. As used herein, “covalently coupled” refers to direct orindirect coupling of two components exclusively through covalent bonds.Direct covalent coupling may involve direct covalent binding between anatom of the β-glucan moiety and an atom of the active moiety.Alternatively, the covalent coupling may occur through a linking groupcovalently attached to the β-glucan moiety, the active moiety, or both,that facilitates covalent coupling of the β-glucan moiety and the activemoiety. Indirect covalent coupling may include a third component suchas, for example, a solid support to which the β-glucan moiety and theactive moiety are separately covalently attached. As used herein,“covalently coupled” and “covalently attached” are used interchangeably.

When present, the linking group can be any suitable organic linkinggroup that allows the β-glucan moiety to be covalently coupled to theactive moiety while preserving the B cell targeting activity of theβ-glucan moiety and an effective amount of activity of the activemoiety.

The linking group can includes a reactive group capable of reacting withthe active moiety to form a covalent bond. Suitable reactive groupsinclude, for example, those discussed in Hermanson, G. (1996),Bioconjugate Techniques, Academic Press, Chapter 2 “The Chemistry ofReactive Functional Groups”, 137-166. For example, the linking group mayreact with a primary amine (e.g., an N-hydroxysuccinimidyl ester or anN-hydroxysulfosuccinimidyl ester); it may react with a sulfhydryl group(e.g., a maleimide or an iodoacetyl), or it may be a photoreactive group(e.g. a phenyl azide including 4-azidophenyl, 2-hydroxy-4-azidophenyl,2-nitro-4-azidophenyl, and 2-nitro-3-azidophenyl).

A chemically active group accessible for covalent coupling to thelinking group includes groups that may be used directly for covalentcoupling to the linking group or groups that may be modified to beavailable for covalent coupling to the linking group. For example,suitable chemically active groups include but are not limited to primaryamines and sulfhydryl groups. Because certain active moieties e.g.,proteins and other peptides may include a plurality of chemically activegroups, certain β-glucan compounds may include a plurality of β-glucanmoieties coupled to an active moiety.

Certain β-glucan compounds may contain chemical associations between theβ-glucan moiety and the active moiety other than covalent coupling. Forexample, a β-glucan compound may include an affinity interaction betweenthe β-glucan moiety and the active moiety. Avidin-biotin affinityrepresents one example of a non-covalent interaction that may beutilized to couple an active moiety and a β-glucan moiety. For example,a biotin molecule may be covalently attached to a proteinaceous activemoiety via one of a number of functional groups present on amino acids(e.g., primary amines or sulfhydryl groups), a β-glucan may be coupledto an avidin molecule by appropriate derivatization of the β-glucanmoiety, and the two moieties may be non-covalently coupled to oneanother through the avidin-biotin affinity interaction. Methods forbiotinylating proteins and linking chemical groups to avidin are wellknown to one of skill in the art. Alternative affinity interactions thatmay be useful for making β-glucan compounds include, for example,antigen/antibody interactions and glycoprotein/lectin interactions.

In some embodiments, the β-glucan compound can include a derivatizedβ-glucan. Generally, β-glucan may be derivatized by, for example,alkylation, adding an amine-containing moiety, side chain modification,or oxidation of primary hydroxyl groups to yield glucuronic acidmoieties.

Thus, the methods described herein can exploit the B cell bindingproperties of the β-glucan compound to directly modulate one or more Bcell biological functions or, alternatively, to deliver an active agentthat can modulate one or more B cell biological functions. Since theβ-glucan compound can modulate B cell biological functions, the β-glucancompounds described herein also can be used to modulate B cell-inducedfunctions or phenomena.

The β-glucan compound can, therefore, be used to target and activate Bcells. Such a method may be used, for example, to induce neutralizing orgrowth-inhibiting antibody for treating an infectious disease or cancer.As another example, FIG. 7 shows the targeted delivery of a therapeuticcompound ERBITUX (Eli Lilly and Co., New York, N.Y. and Bristol-MyersSquibb Co., Princeton, N.J.) to B cells by conjugating the compound to aβ-glucan (IMPRIME PGG).

β-glucan compounds described herein can be used to target immunepotentiating or immune modulating agents to B cells. For example, onemay target an antigen to B cells in order to generate an immune responseto that antigen. The data presented in FIG. 8 demonstrate that IMPRIMEPGG can activate B cells. Calcium flux is an indicator of B cellactivation. FIG. 8 shows that B cells incubated in the presence ofIMPRIME PGG and then stimulated with goat anti-human IgM exhibit anincreased and prolonged period of calcium flux and, therefore, anincreased and prolonged period of activation. This native B cellactivating activity of certain β-glucans may be used to induce a B cellresponse against a particular antigen by coupling an antigen of interestto the B cell-activating β-glucan.

In other cases, the β-glucan may be used to target and inhibit B cellfunction. Such a method may be used, for example, to inhibit antibodyproduction by B cells where B cell function is dysregulated such as, forexample, in autoimmune conditions such as Lupus or rheumatoid arthritis.For example, data presented in FIG. 9 demonstrate that a particulateform of β-glucan, orally administered to a subject, can decrease calciumflux in B cells stimulated with goat anti-human IgM. Thus, activation ofB cells in the β-glucan-treated subjects was inhibited compared to theuntreated control subjects. This B cell inhibiting activity of certainβ-glucans may be used to inhibit the B cell response against aparticular antigen by coupling the antigen of interest to the Bcell-inhibiting β-glucan.

As yet another example, one may target an inducer of immunoglobulinproduction to B cells e.g., a TLR7 agonist or a TLR9 agonist in order togenerate antibody against infectious diseases and cancer. Non-targetedTLR agonists can be poorly tolerated and may have relatively narrowtherapeutic window. Thus, targeting their delivery to B cells as part ofa β-glucan molecule can allow systemic delivery of the TLR agonists withtargeted activity, thereby reducing potential systemic side effects ofsuch agents. Furthermore, β-glucans can act as vaccine adjuvants. Thus,a β-glucan compound that includes a β-glucan moiety and, for example, aPAMP or a DAMP, used in combination with an antigen may enhance immuneresponses and reduce systemic exposure of the DAMP and/or PAMP. Thus, aβ-glucan compound that includes an immunomodulator as an active moiety,when formulated with an antigen in a vaccine, may be retained at a siteof injection and, therefore, may enhance the adjuvant activity of theactive moiety. Such a β-glucan compound may not only be targeted to Bcells, but also may activate B cells near the introduced vaccineantigen, thereby enhancing the presentation of the desired antigen tothe immune system. Moreover, a β-glucan compound that includes a smallmolecule TLR agonist e.g., an imidazoquinoline amine as an active moietycan be retained at the site of administration (e.g., injection orvaccination). Some small molecule immunomodulators can be dispersedsystemically due to their small size and, consequently, be of limitedefficacy at the site of administration. When coupled to a β-glucan asthe active moiety of a β-glucan compound, however, theβ-glucan/immunomodulator β-glucan compound may be of sufficient size sothat the small molecule active moiety is better retained at the site ofadministration.

Certain β-glucan molecules described herein can be used to eliminate Bcells that are neoplastic or that produce autoantibodies. In theseembodiments, a cytotoxic agent may be targeted to B cells by couplingthe cytotoxic active agent to a β-glucan moiety.

In still other cases, β-glucan compounds described herein may be used totarget B cell activators to B cells so that the combination of the Bcell activator and the β-glucan moiety can produce additive orsynergistic effects.

Thus, one or more β-glucan compounds described herein may be formulatedin a composition along with a “carrier.” As used herein, “carrier”includes any solvent, dispersion medium, vehicle, coating, diluent,antibacterial and/or antifungal agent, isotonic agent, absorptiondelaying agent, buffer, carrier solution, suspension, colloid, and thelike. The use of such media and/or agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

As used herein, “pharmaceutically acceptable” refers to a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the β-glucan compound withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition.

One or more β-glucan compounds may be formulated into a pharmaceuticalcomposition. The pharmaceutical composition may be formulated in avariety of forms adapted to a preferred route of administration. Thus, acomposition can be administered via known routes including, for example,oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous,intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g.,intranasal, intrapulmonary, intramammary, intravaginal, intrauterine,intradermal, transcutaneous, rectally, etc.). A pharmaceuticalcomposition can be administered to a mucosal surface, such as byadministration to, for example, the nasal or respiratory mucosa (e.g.,by spray or aerosol). A composition also can be administered via asustained or delayed release.

Such pharmaceutical compositions may be used in methods for treatingcertain conditions where the condition may be treatable throughmodulation of B cell biological function. As noted above, such B cellmodulation can include, depending upon the specific condition,increasing B cell biological function, decreasing B cell biologicalfunction, or targeted killing of B cells.

As used herein, “treat,” “treating,” or variations thereof refer toreducing, limiting progression, ameliorating, or resolving, to anyextent, the symptoms or signs related to a condition. “Ameliorate”refers to any reduction in the extent, severity, frequency, and/orlikelihood of a symptom or clinical sign characteristic of a particularcondition. “Sign” or “clinical sign” refers to an objective physicalfinding relating to a particular condition capable of being found by oneother than the patient. “Symptom” refers to any subjective evidence ofdisease or of a patient's condition.

The methods may be used to treat a condition prophylactically ortherapeutically. As used herein, “prophylactic” and variations thereofrefer to a treatment that limits, to any extent, the development and/orappearance of a symptom or clinical sign of a condition. In many cases,prophylactic treatment can occur before any symptom or clinical sign ofthe condition is apparent. As used herein, “therapeutic” and variationsthereof refer to a treatment that ameliorates one or more existingsymptoms or clinical signs associated with a condition. “Treatment”refers to a course of action or one of a series of actions for treatinga condition.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the one or more β-glucan compounds intoassociation with a carrier that constitutes one or more accessoryingredients. In general, a formulation may be prepared by uniformlyand/or intimately bringing the active compound into association with aliquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

One or more β-glucan compounds may be provided in any suitable formincluding but not limited to a solution, a suspension, an emulsion, aspray, an aerosol, or any form of mixture. The composition may bedelivered in formulation with any pharmaceutically acceptable excipient,carrier, or vehicle. For example, the formulation may be delivered in aconventional topical dosage form such as, for example, a cream, anointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion,and the like. The formulation may further include one or more additivesincluding such as, for example, an adjuvant, a skin penetrationenhancer, a colorant, a fragrance, a flavoring, a moisturizer, athickener, and the like.

Generally, the methods include administering to a subject in need oftreatment an amount of a β-glucan compound described herein effective tomodulate at least one B cell biological function.

The amount of β-glucan compound administered can vary depending onvarious factors including, but not limited to, the specific β-glucancompound or compounds being administered, the weight, physicalcondition, and/or age of the subject, and/or the route ofadministration. Thus, the absolute weight of β-glucan compound includedin a given unit dosage form can vary widely, and depends upon factorssuch as the species, age, weight and physical condition of the subject,as well as the method of administration. Accordingly, it is notpractical to set forth generally the amount that constitutes an amountof β-glucan compound effective for all possible applications. Those ofordinary skill in the art, however, can readily determine theappropriate amount with due consideration of such factors.

In some embodiments, the methods of the present invention includeadministering sufficient β-glucan compound to provide a dose of, forexample, from about 100 ng/kg to about 50 mg/kg to the subject, althoughin some embodiments the methods may be performed by administering theβ-glucan compound in a dose outside this range. In some of theseembodiments, the method includes administering sufficient β-glucancompound to provide a dose of from about 10 μg/kg to about 5 mg/kg tothe subject, for example, a dose of from about 100 μg/kg to about 1mg/kg.

Alternatively, the dose may be calculated using actual body weightobtained just prior to the beginning of a treatment course. For thedosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184.

In some embodiments, the methods of the present invention may includeadministering sufficient β-glucan compound to provide a dose of, forexample, from about 0.01 mg/m² to about 1000 mg/m² such as, for example,a dose of about 500 mg/m². In some cases, a β-glucan compound can beadministered at one initial dose of, for example, 500 mg/m², thenfollowed by subsequent lesser doses such as, for example, 250 mg/m².

In some embodiments, β-glucan compound may be administered, for example,from a single dose to multiple doses per week, although in someembodiments the methods of the present invention may be performed byadministering β-glucan compound at a frequency outside this range. Incertain embodiments, β-glucan compound may be administered from aboutonce every 12 weeks, once every eight weeks, once every four weeks, oronce every week.

As used herein, the term “and/or” means one or all of the listedelements or a combination of any two or more of the listed elements; theterm “comprises” and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims; unlessspecifically stated otherwise, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one; and recitations ofnumerical ranges by endpoints include all numbers subsumed within thatrange (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiment can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

FIG. 2 demonstrates that some β-glucans, such as IMPRIME PGG andLaminarin, bind directly to human B cells in whole blood and enrichedperipheral blood mononuclear cell (PBMC) population in aconcentration-dependent manner. This ability was evaluated by comparingthe levels of B cell-bound β-glucans detected by BfD IV, a monoclonalantibody (MAb) specific for β-1,3/1,6 glucans (U.S. Pat. No. 6,294,321).The histograms in FIG. 2 show the median fluorescence intensity (MFI) ofBfD IV staining of various concentrations of β-glucans on B cells inwhole blood (A) and enriched PBMC (B and C).

β-Glucan Binding Methodology:

A) Binding in Whole Blood:

Blood was collected from a healthy donor with 10 Units/mL Heparin. 100μL of blood was mixed with IMPRIME PGG concentrations and incubated at37° C. for 60 minutes. Cells were washed with 2 mL of PBS andsupernatant removed by aspiration. Cells were then stained with 20 μL ofBfD IV (U.S. Pat. No. 6,294,321) for 30 minutes before washing againwith PBS. 10 μL of goat anti-mouse IgM FITC added to stain for BfD IVpositive cells, while 10 μL of anti-CD19 APC was added to stain the Bcell population for FACS analysis. Cells were incubated for 30 minutesat room temperature before washing with PBS. The red cells in the bloodwere lysed by the addition of 2 mL of BD Lyse and fixed with 2%paraformadehyde before analysis on an LSR II flow cytometer. Analysiswas performed using FlowJo software. Results are shown in FIG. 2A.

B) Binding in PBMC:

Enriched PBMC were resuspended at 1×10⁶ cells/mL in RPMI 1640supplemented with 10% serum. The β-glucans (IMPRIME PGG (B) andlaminarin (C)) at various hexose concentration were added to the cellsand incubated in a 37° C., 5% C02 humidified incubator for two hours.After incubation, cells were washed twice with FACS buffer (HBSSsupplemented with 1% FBS and 0.1% sodium azide) to remove any unboundβ-glucan, and subsequently treated with Fc block. After the Fc blockstep, cells were stained with the monoclonal antibody BfD IV (U.S. Pat.No. 6,294,321) for 30 minutes at 4° C. and washed twice with cold FACSbuffer. Cells were then incubated with FITC-conjugated F(ab′)2 goatanti-mouse IgM for 30 minutes at 4° C. and washed once with cold FACSbuffer before fixing with 1% paraformaldehyde. Events were collected ona LSRII flow cytometer and analysis was performed using FlowJo software.Results are shown in FIG. 2B (IMPRIME PGG) and FIG. 2C (laminarin).

Example 2

This example demonstrates that IMPRIME PGG binds to complement receptor2, (CR2; aka CD21) on B cells. FIG. 3A shows that CR2 is abundantlyexpressed on human B-cells, but not on monocytes, neutrophils, or NKcells.

CR2 Staining Methodology:

Enriched PMNs, B cells, monocytes, or NK cells were resuspended at 1×10⁶cells/mL in FACS buffer (HBSS supplemented with 1% FBS and 0.1% sodiumazide were stained with PE-conjugated anti-CR2 mouse Ab (LT-21) or mouseIgG1 isotype control and subsequently analyzed by flow cytometry.Results are shown in FIG. 3A.

β-Glucan Binding and CR2 Blocking Methodology:

Whole blood or PBMC were pre-incubated with specific receptor blockingantibodies or the relevant isotype controls at 4° C. for 30-45 minutesbefore addition of IMPRIME PGG at 100 μg/mL. Binding to B cells wasperformed as described in Example 1. Clone 1048, the mouse mAb againstCR2, and mouse IgG1 isotype control was used at 10 μg/mL/1×10⁶ cells.Results are shown in FIG. 3B (Whole Blood) and FIG. 3C (PBMC).

Example 3

This example demonstrates that IMPRIME PGG not only binds to normalhuman cells, but also to B cell tumor lines. The histograms in FIG. 4show that a) human B-cell tumor lines, Daudi and Raji express CR2, andb) IMPRIME PGG bound to these tumor lines in a concentration-dependentmanner.

Staining of CR2 and Binding of IMPRIME PGG to B-Cell Lines Methodology:

Daudi or Raji cells, resuspended at 1×10⁶ cells/mL in FACS buffer (HBSSsupplemented with 1% FBS and 0.1% sodium azide were stained withPE-conjugated anti-CR2 (LT-21) mouse mAb or mouse IgG1 isotype controland subsequently analyzed by flow cytometry as described in Example 1.Results are shown in FIG. 4A and FIG. 4B.

Example 4

This example demonstrates that conjugating bovine serum albumin (BSA) toIMPRIME PGG enhances its binding capacity to human B cells. Thehistogram in FIG. 5 shows that the mean fluorescent intensity (MFI) ofBfD IV staining on IMPRIME PGG-BSA conjugate (BT-1110)-treated B cellsis higher at the tested concentration (50 μg/mL) compared to parentcells treated with unconjugated IMPRIME PGG.

The binding of the conjugated and unconjugated β-glucans, subsequentstaining with BfD IV (U.S. Pat. No. 6,294,321), and flow cytometry wereperformed as described in Example 1. Results are shown in FIG. 5.

Example 5

This example demonstrates that derivatization of IMPRIME PGG enhancesits binding capacity to human B cells. The histogram of FIG. 6 showsthat the mean fluorescent intensity (MFI) of BfD IV staining on B cellstreated with a benzyl amine derivative of IMPRIME PGG (BT-1222) ishigher at the tested concentration (10 μg/mL) compared to parent cellstreated with underivatized IMPRIME PGG.

Example 6

Whole blood was incubated with 10 μg/mL IMPRIME PGG, ERBITUX (Eli Lillyand Co., New York, NY and Bristol-Myers Squibb Co., Princeton, N.J.),ERBITUX-IMPRIME PGG conjugate, or citrate buffer (as a control) for 30minutes at 37° C. Cells were washed twice with PBS before staining withthe β-glucan specific antibody BfD IV (U.S. Pat. No. 6,294,321). Cellswere washed again and stained with an antibodies specific for human IgGto detect ERBITUX and CD19 to label B cells. Cells were analyzed on andLSRII flow cytometer and the groups were compared by gating on CD19⁺Bcells. In the left panel, cells treated with either IMPRIME PGG or theERBITUX conjugate were positive for β-glucan but only the conjugatetreated group had an increase in anti-human IgG binding in the rightpanel showing the conjugated β-glucan was able to target the CD19⁺ Bcell population. Results are shown in FIG. 7.

Example 7 PBMC Staining With 3 μM Fura Red and Fluo-4

FURA RED and FLUO-4 (each from Invitrogen, Life Technologies Corp.,Carlsbad, Calif.) calcium stain was added to serum free RPMI medium to afinal concentration of 3 μM for each dye, then incubated at 37° C. for30 minutes. PBMCs mixed 1:1 with media containing 10% FCS and incubatedan additional 10 minutes. The cells were washed 2× with media containing10% FCS. Cells were resuspended in RPMI with 10% human serum.

The resuspended cells were incubated with or without the addition of 100μg/mL IMPRIME PGG at 37° C. for one hour. The monoclonal antibody CD20APC (BioLegend, Inc., San Diego, Calif.) was added to each tube for 10minute incubation before washing 2× with RPMI containing 10% FCS. Cellswere resuspended in RPMI with 10% FCS for FACS analysis. Calcium fluxwas induced by stimulating the B cells with goat anti-human IgM at 2.6μg/mL. Results are shown in FIG. 8 and demonstrate the ability ofIMPRIME PGG-treated B cells to have a larger and more sustained calciumflux, which corresponds to increased B cell responsiveness to antigenchallenges and/or B cell receptor stimulation.

Example 8

Blood was drawn from untreated donors (n=15) or donors (n=12) treatedwith particulate oral β-glucan (U.S. Pat. No. 7,981,447) at dosagesranging from 100 mg/day to 1000 mg/day. PBMCs were isolated from bloodusing a ficoll gradient.

The isolated PBMCs were treated with 6 μM FURA RED and 3 μM FLUO-4 (eachfrom Invitrogen, Life Technologies Corp., Carlsbad, Calif.) in PBS for45 minutes at 37° C. The cells were washed 2' with PBS and stained withanti-CD20 APC (BioLegend, Inc., San Diego, Calif.) for 20 minutes atroom temperature. Cells were washed 2× and resuspended in RPMIcontaining 2.5% FCS for FACS analysis. Calcium flux was induced bystimulating the B cells with goat anti-human IgM at 10 μg/mL, 2 μg/mL,or 0.4 μg/mL.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A method comprising: administering to a subject an amount of aβ-glucan compound effective to bind to B cells and modulate at least onebiological function of the B cells, the composition comprising: aβ-glucan moiety; and an active moiety coupled to the β-glucan moiety,the active moiety comprising a danger-associated molecular pattern.2-18. (canceled)
 19. The method of claim 1 wherein the β-glucan moietycomprises a soluble β-glucan or a particulate β-glucan.
 20. The methodof claim 1 wherein the β-glucan moiety comprises a derivatized β-glucan.21. The method of claim 1 wherein the β-glucan moiety is isolated fromyeast.
 22. The method of claim 21 wherein the yeast comprisesSaccharomyces cerevisiae.
 23. The method of claim 1 wherein thedanger-associated molecular pattern comprises a heat-shock protein,high-mobility group box 1 protein, a protein generated following tissueinjury, a hyaluronan fragment, ATP, uric acid, heparin sulfate or DNA.24. The method of claim 1 wherein the β-glucan moiety and the activemoiety are coupled through a covalent linkage.
 25. The method of claim 1wherein the β-glucan moiety and the active moiety are coupled through anaffinity linkage.
 26. The method of claim 1 wherein at least onebiological function of the B cells comprises making an immunoglobulininvolved in cancer therapy.
 27. The method of claim 1 wherein modulatingat least one biological function of the B cells comprises making animmunoglobulin involved in infectious disease treatment.
 28. A β-glucancompound comprising: a β-glucan moiety; and an active moiety coupled tothe β-glucan moiety, the active moiety comprising a danger-associatedmolecular pattern.
 29. The β-glucan compound of claim 28 wherein theactive moiety comprises a heat-shock protein, high-mobility group box 1protein, a protein generated following tissue injury, a hyaluronanfragment, ATP, uric acid, heparin sulfate or DNA.
 30. The β-glucanmoiety of claim 28 wherein the β-glucan moiety is isolated from yeast.31. The β-glucan moiety of claim 30 wherein the yeast comprisesSaccharomyces cerevisiae.
 32. The β-glucan moiety of claim 28 whereinthe β-glucan moiety and the active moiety are coupled through a covalentlinkage or an affinity linkage.
 33. A pharmaceutical compositioncomprising: a β-glucan compound comprising: a β-glucan moiety; and anactive moiety coupled to the β-glucan moiety, the active moietycomprising a danger-associated molecular pattern; and a pharmaceuticallyacceptable carrier.
 34. The pharmaceutical composition of claim 33wherein the active moiety comprises a heat-shock protein, high-mobilitygroup box 1 protein, a protein generated following tissue injury, ahyaluronan fragment, ATP, uric acid, heparin sulfate or DNA.
 35. Thepharmaceutical composition of claim 33 wherein the β-glucan moiety isisolated from yeast.
 36. The pharmaceutical composition of claim 35wherein the yeast comprises Saccharomyces cerevisiae.
 37. Thepharmaceutical composition of claim 33 wherein the β-glucan moiety andthe active moiety are coupled through a covalent linkage or an affinitylinkage.